IL-6 mRNA Levels Research Articles

Organophosphate Insecticide Malathion Induces Alzheimer's Disease-Like Cognitive Impairment in Mice: Evidence of the Microbiota-Gut-Brain Axis.

Evidence suggests that exposure to organophosphate pesticides increases the risk of neurodegenerative diseases, but the mechanisms remain unclear. This study investigated the effects of malathion on Alzheimer's disease (AD)-like symptoms at environmentally relevant concentrations using wild-type (WT) and APP/PS1 transgenic mouse models. Results showed that malathion exposure induced AD-like cognitive impairment, amyloid-β (Aβ) accumulation, and neuroinflammation in WT mice, with worsened symptoms in APP/PS1 mice. Mechanistic studies revealed that malathion induced AD-like gut microbiota dysbiosis (reduced Lactobacillus and Akkermansia, and increased Dubosiella), causing gut barrier impairment and tryptophan metabolism disruptions. This resulted in a significant increase in indole derivatives and activation of the colonic aryl hydrocarbon receptor (AhR), promoting the kynurenine (KYN) pathway while inhibiting the serotonin (5-HT) pathway. Increased neurotoxic KYN metabolites (3-hydroxykynurenine and quinolinic acid) triggered gut and systemic inflammation, upregulating hippocampal IL-6 and IL-1β mRNA levels and thereby causing neuroinflammation. Gut tryptophan metabolism disruptions caused hippocampal neurotransmitter imbalances, reducing the levels of 5-HT and its derivatives. These effects promoted AD progression in both WT and APP/PS1 mice. This study highlights the crucial role of the microbiota-gut-brain axis in AD-like cognitive impairment induced by malathion exposure, providing insights into the neurodegenerative disease risks posed by organophosphate pesticides.

Anti-inflammatory effects of eupatilin on Helicobacter pylori CagA-induced gastric inflammation.

Eupatilin, a flavone isolated from Artemisia species, exerts anti-inflammatory, anti-oxidative, and anti-neoplastic activities. However, the effects of eupatilin on H. pylori-associated gastritis remain unclear. Thus, this study aimed to investigate the anti-inflammatory effects of eupatilin on gastric epithelial cells infected with cytotoxin-associated gene A (CagA)-positive Helicobacter pylori. AGS human gastric carcinoma cells were infected with a CagA-positive H. pylori strain and then treated with 10, 50, or 100 ng of eupatilin. After 24 h, the expression levels of CagA, phosphoinositide 3-kinase 1 (PI3K), nuclear factor (NF)-κB, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the cell lysates were measured using western blotting, and the mRNA levels of IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 were measured using real-time polymerase chain reaction. CagA translocation into AGS cells resulted in an elongated cell morphology, which was significantly suppressed by eupatilin treatment in a dose-dependent manner. Immunofluorescence staining for anti-CagA showed that eupatilin treatment dose-dependently inhibited CagA expression in the H. pylori-infected AGS cells. H. pylori infection increased the levels of pro-inflammatory cytokines including IL-1β, TNF-α, IL-6, IL-8, and MCP-1, and eupatilin treatment significantly reduced the levels of these cytokines in a dose-dependent manner. Additionally, eupatilin treatment dose-dependently suppressed the expression of PI3K and NF-κB. Eupatilin treatment demonstrated anti-inflammatory effects on CagA-positive H. pylori-infected gastric epithelial cells by inhibiting CagA translocation, thereby suppressing the NF-κB signaling pathway. These results suggest that eupatilin plays a protective role against CagA-positive H. pylori-induced gastritis.

Exploration of the Active Components and Mechanism of Jiegeng (Platycodonis Radix) in the Treatment of Influenza Virus Pneumonia Through Network Pharmacology Analysis and Experimental Verification.

This study aimed to explore the pathogenesis of platycodin D and luteolin, which are both active components in Jiegeng (Platycodonis Radix), in the treatment of influenza virus pneumonia through network pharmacology analysis combined with experimental verification. The bioactive components of Jiegeng (Platycodonis Radix) were screened by TCMSP and literature mining, and the results were standardized via the UniProt database. The action targets for the disease were identified from databases including OMIM, GeneCards, TTD, DisGeNET, and PharmGKB. Then, the visualized key target regulatory network and protein-protein interaction (PPI) network for the active components were established using Cytoscape3.7.1 software. The findings were illustrated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The intervention concentrations of platycodin D and luteolin were screened by the CCK8 method, and the important signaling pathways of platycodin D and luteolin for treating influenza virus pneumonia were verified by RT-qPCR and Western blot tests. From data mining, 89 common drug-disease targets were screened out, and five major active components of Jiegeng (Platycodonis Radix), including platycodin D and luteolin, were obtained. Besides, 11 therapeutic targets including IL-17, IL-6, TNF-α, JUN, and MAKP1 were identified by PPI network analysis. GO and KEGG enrichment analyses showed that the pathways most related to the mechanisms of Jiegeng (Platycodonis Radix) against influenza virus pneumonia included the TNF and IL-17 signaling pathways and apoptosis. Invitro experiments demonstrated that the model group exhibited a notable elevation in mRNA levels of IL-6, IL-17, TNF-α, JUN, MAPK1, and the IL-17/-acting protein ratio, as compared to the control group (p < 0.05). In contrast to the model group, the IL-6, IL-17, TNF-α, JUN, MAPK1 mRNA expression levels, and the IL-17 protein ratio in both the platycodin D group and luteolin group were considerably decreased (p < 0.05). Combined with network pharmacology and experimental verification, this study revealed that platycodin D and luteolin in Jiegeng (Platycodonis Radix) may treat influenza virus pneumonia by regulating inflammation through the IL-17 signaling pathway.

Motor, mood, and memory impairments persist during remission periods in chronic colitis and are influenced by neuroinflammation and sex.

Ulcerative colitis is a chronic pathology characterized by relapsing-remitting phases of intestinal inflammation. Additionally, some patients develop neuropsychiatric disorders, such as depression and anxiety, or cognitive deficits. We aimed to investigate whether the development of chronic colitis elicits memory, locomotion, and mood impairments. It further examined whether these impairments are influenced by the relapsing-remitting phases of the colitis or by sex. Here, we used a chronic colitis model in male and female rats, induced with sodium dextran sulfate, mirroring the phases of human ulcerative colitis. Our results revealed that the severity of colitis was slightly higher in males than females. Chronic colitis triggered motor and short-term memory deficits and induced anxiety- and depression-like behaviors that remained throughout the development of the disease. There are also sex differences under control or inflammatory conditions. Therefore, in both situations, females compared to males displayed: (i) slightly lower locomotion, (ii) increased anxiety-like behaviors, (iii) similar depression-like behaviors, and (iv) similar short-term memory deficit. Additionally, under control conditions, the mRNA levels of IL-1β, IL-6, and TNF-α were higher in the female hippocampus. In both sexes, when chronic colitis was established, the neuroinflammation was evidenced by increased mRNA levels of these three cytokines in the hippocampus and in the motor and prefrontal cortices. Interestingly, this neuroinflammation was slightly greater in males. In summary, we show that the development of chronic colitis caused persistent behavioral abnormalities, highlighting sex differences, and that could be a consequence, at least in part, of the increase in IL-1β, IL-6, and TNF-α in the brain.

Scutellarein ameliorates dextran sulfate sodium-induced ulcerative colitis by inhibiting colonic epithelial cell proinflammation and barrier disruption.

Scutellarein (Scu) is a natural occurring flavonoid found in multiple traditional Chinese medicines such as Oroxylum indicum (L.) Kurz and Scutellaria baicalensis, with various pharmacological activities including anti-inflammation, anti-oxidation and myocardial protection. Here, we investigated the therapeutic efficacy of Scu on ulcerative colitis (UC) and the underlying mechanism. Efficacy of Scu on UC was evaluated in dextran sulfate sodium (DSS) induced colitis mouse model. Inflammation in colonic tissues was assessed by myeloperoxidase activity assay and RT-qPCR. Barrier proteins expression was examined using immunostaining and Western blot. IL-1β-treated HT-29 cells was used for mechanical investigation. Gavage of Scu significantly decreased the DAI score, improved colon shortening, ameliorated the pathological score in DSS-treated mice with better efficacy than the positive drug, 5-aminosalicylic acid. Scu also inhibited the expression levels of cytokines (Il-1β, Tnf-α, Il-1α, Il-6, and Cxcl1) as well as barrier proteins (E-cadherin, Occludin, and ZO-1) in colon tissues of DSS mice. In intestinal epithelial HT-29 cells, Scu attenuated the IL-1β-downregulated expression levels of E-cadherin, occludin, and ZO-1, while reduced IL-1β-upregulated IL-6 and IL-8 mRNA levels. Moreover, Scu inhibited the phosphorylation and nuclear translocation of NF-κB and suppression of NF-κB phosphorylation abolished IL-1β-disrupted epithelial barrier integrity and IL-1β-upregulated proinflammatory mediators expression in HT-29 cells. These data demonstrate that Scu is an efficacious therapeutic agent to treat UC. Inhibition of inflammatory responses and maintenance of epithelial barrier integrity through NF-κB signaling pathway underlines Scu therapeutic effect on UC.

Indigo alleviates psoriasis through the AhR/NF-κB signaling pathway: an in vitro and in vivo study.

Psoriasis is a chronic inflammatory skin disease. A strong association between the AhR/ NFκB axis and the inflammatory response in psoriasis. Indigo (IDG) has demonstrated significant anti-inflammatory properties. This study aimed to assess the anti-psoriatic efficacy of IDG while investigating the underlying mechanisms involved. In the in vitro experiments, cell viability was assessed using the CCK-8. qRT-PCR was employed to measure the mRNA levels of NF-κB, TNF-α, IL-1β, AhR, and CYP1A1. Western blotting was conducted to examine alterations in cytoplasmic and nuclear AhR protein levels. Additionally, an IDG nanoemulsion (NE) cream was prepared for the in vivo experiments. A psoriasis-like skin lesion mice model was induced using IMQ (62.5 mg/day for 7 days). The severity of psoriasis was evaluated using PASI, and skin lesions were scored while epidermal thickness was assessed via HE staining. The expression of inflammatory markers, including IL-6, IL-13, IL-17A, MCP-1, and TNF-α, was detected in skin lesions using Luminex. The levels of CYP1A1, p65, and p-p65 proteins were determined by Western blotting. LPS stimulation significantly elevated TNF-α, IL-6, and NF-κB mRNA levels, which were notably reduced by IDG treatment. Additionally, IDG significantly enhanced the expression of AhR and CYP1A1 mRNA. Further investigation revealed that IDG facilitated AhR translocation from the cytoplasm to the nucleus. In the IMQ-induced psoriasis-like mouse model, IDG NE substantially ameliorated the severity of skin lesions. Moreover, IDG NE treatment reduced the upregulation of inflammatory cytokines such as IL-6, IL-17A, MCP-1, and TNF-α in IMQ-induced skin lesions. It was also observed that IDG NE treatment increased CYP1A1 protein expression while inhibiting p65 and p-p65 protein expression. IDG emerges as a promising treatment for psoriasis, demonstrating effective therapeutic outcomes. Its mechanism of action is likely linked to the modulation of the AhR/NFκB signaling pathway.

Role of AhR-Hsp90-MDM2-mediated VDR ubiquitination in PM2.5-induced renal toxicity

Background and objectivesThe kidney is a primary target for the accumulation of particulate matter (PM2.5). This study aimed to investigate PM2.5-induced renal toxicity mechanisms, focusing on the aryl hydrocarbon receptor (AhR)-Hsp90-MDM2 axis and its impact on vitamin D receptor (VDR) ubiquitination. MethodsPM2.5's role in activating the AhR and its downstream pathways was investigated using in vitro and in vivo models. Renal damage and therapeutic effects in PM2.5-exposed and paricalcitol-treated mice were evaluated using weight measurements, histopathology, and scanning electron microscopy. AhR, Hsp90, and VDR localization and expression in renal cells were assessed using FISH and Western blot. Protein interactions were examined using co-immunoprecipitation. Differentially expressed gene (DEG) analysis of GEO datasets was used to identify related proteins and genes. ResultsPM2.5 exposure caused significant renal damage in mice, including increased serum creatinine, albuminuria, and histopathological deterioration, which were alleviated by paricalcitol. PM2.5 induced the nuclear translocation of AhR and Hsp90 and reduced nuclear VDR expression; paricalcitol reversed these effects. Immunohistochemistry confirmed these findings. PM2.5 upregulated the NLRP3/caspase-1/IL-1β/IL-18 axis, which was reversed by paricalcitol treatment. Inhibition of Hsp90 increased nuclear VDR expression through MDM2 mediation. DEG analysis identified VDR-regulated genes; PM2.5 increased the mRNA levels of IL-6, IL-2, and CXCL8, which were downregulated by Hsp90 and MDM2 inhibitors, with VDR agonists further decreasing these levels. ConclusionThis study reveals a novel mechanism of PM2.5-induced renal toxicity through the AhR-Hsp90-MDM2 axis, promoting VDR ubiquitination and degradation and increasing inflammation. These findings provide a foundation for future studies and lay the groundwork for developing targeted interventions to mitigate the public health impact of PM2.5 exposure.

Effect of Endurance Exercise Training on Gut Microbiota and ER Stress

Regular exercise as part of one’s lifestyle is well-recognized for its beneficial effect on several diseases such as cardiovascular disease and obesity; however, many questions remain unanswered regarding the effects of exercise on the gut environment. This study aimed to investigate the impact of long-term endurance exercise on modulating inflammation and endoplasmic reticulum (ER) stress. Fifteen-week-old male Sprague-Dawley (SD) rats were subjected to six months of endurance treadmill training, while age-matched controls remained sedentary. Results showed that IL-6 mRNA levels in colon tissues were significantly higher in the exercise group compared to the sedentary group. Exercise activated a significant ER stress-induced survival pathway by increasing BiP and phosphorylation of eIF2α (p-eIF2α) expressions in the liver and colon, while decreasing CHOP in the liver. Gene expressions of MUC2, Occludin, and Claudin-2 were increased in the colon of the exercise group, indicating enhanced intestinal integrity. Furthermore, the data showed a positive correlation between microbiota α-diversity and BiP (r = 0.464~0.677, p &lt; 0.05). Populations of Desulfovibrio C21 c20 were significantly greater in the exercise group than the sedentary group. Additionally, predicted functions of the gut microbial community in terms of enzymes and pathways supported the enhancement of fatty-acid-related processes by exercise. These findings suggest that prolonged endurance exercise can affect the colon environment, which is likely related to changes in inflammation, ER stress, mucin layers and tight junctions, associated with modifications in the gut microbiome.

Dietary Bacteriophage Administration Alleviates Enterotoxigenic Escherichia coli-Induced Diarrhea and Intestinal Impairment through Regulating Intestinal Inflammation and Gut Microbiota in a Newly Weaned Mouse Model

This study aimed to investigate the effects of dietary bacteriophage administration on diarrhea and intestinal impairment induced by enterotoxigenic Escherichia coli (ETEC) in a newly weaned mouse model. Forty-four newly weaned C57BL/6 mice were divided into four treatment groups, where they were provided either the control diet or the bacteriophage-supplemented diet, with or without ETEC infection. The results show that the bacteriophage administration resulted in increased body weight, decreased diarrhea score, and improved jejunal histopathology in ETEC-infected mice. The bacteriophage administration enhanced the intestinal barrier function of the ETEC-infected mice, as indicated by the reduced serum DAO level and the increased expression of Claudin-1, Occludin, and ZO-1 at both the mRNA and protein levels in the jejunum. Also, the bacteriophage administration resulted in a decrease in serum TNF-α and IL-1β levels, a down-regulation of TNF-α and IL-6 mRNA levels in the jejunum, and the inhibition of jejunal TLR-4/NF-κB pathway activation induced by ETEC infection. Moreover, the bacteriophage administration increased the levels of acetic acid, propionic acid, butyric acid, and total short-chain fatty acids in the caecum content. The bacteriophage administration increased the Shannon index, increased the abundance of Bacteroidota and Muribaculaceae, and decreased the abundance of Verrucomicrobiota and Akkermansiaceae in the colon contents of the ETEC-infected mice. Spearman’s correlation analysis indicates that the protective effects of bacteriophage on ETEC-induced intestinal impairment, inflammation, and intestinal barrier function are associated with regulating the abundance of Bacteroidota and Muribaculaceae in the colon contents of mice. Collectively, bacteriophage administration alleviates ETEC-induced diarrhea and intestinal impairment through regulating intestinal inflammation and gut microbiota in newly weaned mice.

Chlorogenic Acid Plays an Important Role in Improving the Growth and Antioxidant Status and Weakening the Inflammatory Response of Largemouth Bass (Micropterus salmoides).

This experiment evaluated the function of chlorogenic acid (CGA) in the growth, health status, and inflammation of largemouth bass (Micropterus salmoides). Over eight weeks, CGA supplementation was designed at five levels: 0, 60, 120, 180, and 240 mg/kg. The 180 and 240 mg/kg CGA-supplemented groups showed significant improvements in the FBW, SGR, and WGR compared to the control group (0 mg/kg) (p < 0.05). All the CGA-supplemented groups exhibited a significant reduction in the FCR (p < 0.05), with the 180 mg/kg CGA group showing the lowest FCR. Nonetheless, there were no appreciable differences in the plasma concentrations of TP, ALT, or AST among the treatments (p > 0.05). Compared to the control group, the 180 mg/kg CGA group exhibited significantly lower TC and TG levels (p < 0.05). The ALP levels showed no significant differences from the control group (p > 0.05). In terms of antioxidant parameters, CGA supplementation considerably reduced the MDA content (p < 0.05) and increased the GSH levels, while decreasing the CAT, SOD, and GPx activity levels Meanwhile, CGA supplementation resulted in reduced mRNA levels of SOD, CAT, Nrf2, Keap1, and NF-κB compared to the control group. In contrast, the mRNA levels of GPx, IL-8, TLR2, and RelA were elevated in the liver. Our findings indicated that CGA supplementation improved the growth performance and antioxidant status and weakened the inflammatory response of largemouth bass. These findings suggest that CGA could be a valuable dietary supplement for enhancing the health and growth of this species.

Mycoplasma pneumoniae regulates the expression of GP130 in lung epithelial cells through apoptosis and TLR4/ NF-κB pathway during infection

In previous study, lower levels of serum GP130 were reported in children with MPP. GP130 is an important signal transducer, the down regulation of which may influence host immune responses. In this study, we aimed to analyze the regulatory mechanism of GP130 during MP infection. Firstly, the mRNA and protein levels of GP130 both decrease and then increase with increasing multiplicity of infection (MOI: 1 to 40) of MP. The lowest levels of GP130 were detected at MOI of 5. Then, heat treated MP but not trypsin treated MP or MP extracted proteins show regulatory effect to the expression of GP130. These indicate that the down regulation of GP130 is related to protein mediate adhesion process of MP. Gene expression analysis revealed that MP affected apoptosis and the TLR4 pathway in infected cells, and the mRNA level of IL-6 was correlated with that of GP130. Further, Z-VAD-FMK (pan-caspase inhibitor) can suppress the apoptosis induced by MP infection and restore GP130 at protein level. Further studies revealed that MP infection promoted TLR4 internalization but did not activate the NF-κB pathway. The levels of surface TLR4 showed correlation with the transcription of IL-6 and GP130. TAK242 (TLR4 inhibitor) and PS341 (proteasome inhibitor) can restore the decreased transcription of GP130, both of which were able to promote NF-κB pathway activation in MP-infected cells. These suggested that the regulation of TLR4/NF-κB pathway and induced apoptosis post MP infection are involved in the down-regulation of GP130 at transcription and protein levels, respectively.

Siwu tablet attenuates high fructose-induced glomerular podocyte senescence in rats through increasing Nup155 to promote INO80 mRNA nuclear export

Ethnopharmacological relevanceSiwu tablet (SWT), derived from a traditional Chinese medicinal formula named Siwu decoction, is widely used for blood deficiency syndrome. Siwu decoction and its derived formulas have been proven to improve renal anemia and prevent senescence. Whether SWT prevents glomerular podocyte senescence and the underlying molecular mechanism remains unknow. Aim of the studyTo elucidate the protective effect and possible mechanism of SWT on glomerular podocyte senescence. Material and methodsLiquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to characterize components of SWT. Male Sprague-Dawley rats were given 10% fructose drinking water for 16 weeks. SWT (810 and 1620 mg/kg) was administered orally for the last 8 weeks. The assays of senescence-associated beta-galactosidase (SA-β-gal) staining, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot as well as enzyme linked immunosorbent assay were performed to evaluate rat glomerular podocyte senescence. The mRNA and protein levels of nucleoporin 155 (Nup155) and inositol requiring mutant 80 (INO80) in rat glomeruli were detected by qRT-PCR, Western blot and immunofluorescence. Foot processes and nuclear pore complexes (NPCs) of rat glomerular podocytes were visualized by transmission electron microscopy. ResultsOne hundred and fifty-nine components were preliminarily identified in SWT. The results of animal experiments showed that SWT decreased the activity of SA-β-gal, protein levels of p16, p21, p53 and phosphorylated histone H2AX (γ-H2AX), and mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in glomeruli of high fructose-fed rats. As expected, SWT increased renal cortex erythropoietin mRNA expression and serum erythropoietin concentration in this animal model. SWT reduced urine albumin-to-creatinine ratio and serum levels of uric acid, creatinine and blood urea nitrogen, and recovered glomerular structure injury in high fructose-fed rats. It up-regulated mRNA and protein levels of Nup155 and the number of podocyte NPCs, and subsequently reinforced mRNA nuclear export and protein expression of INO80 in rat glomeruli under high fructose stimulation. ConclusionsSWT ameliorates glomerular podocyte senescence in high fructose-fed rats possibly by increasing Nup155 to promote INO80 mRNA nuclear export.

Three hepcidins from the spotted knifejaw (Oplegnathus punctatus) promote antimicrobial activity via TLR/NFκB pathway

Hepcidin belongs to a class of small cationic antimicrobial peptides rich in cysteine. It is synthesized by liver and is widely involved in host antimicrobial, antiviral and other immune responses. We identified and characterized three hepcidin genes (OpHep1, OpHep2 and OpHep3) in spotted knifejaw. All the OpHeps shared high identities with hepcidins in other teleost, containing alpha helix and β-sheets. Three OpHeps were all detected in healthy tissues, with the abundant expression in liver. They were significantly increased after Vibrio harveyi infection in the six immune-relevant tissues (liver, kidney, spleen, gill, skin and intestine). OpHeps knockdown in spotted knifejaw liver cells affected the mRNA levels of inflammation-related genes, including il1β, il6, il8, and nfκb. Further, the recombinant hepcidin proteins were effective in suppressing the growth of both Gram-negative and Gram-positive bacteria. To identify the function of OpHeps in vivo, we performed the overexpression of three OpHeps in zebrafish, and found OpHeps could significantly induce immune-related genes expression in transgenic zebrafish, including myd88, il10, il21, il16, tlr1, tlr3 and lysozyme. When infected with V. harveyi, OpHeps transgenic zebrafishes had a higher survival rate than wild-type zebrafishes. The expression of myd88, il10, il8, il1β, nfκb and lysozyme were all significantly up-regulated in transgenic fishes during bacterial infection. In summary, these results indicated that hepcidin could protect fish fight against pathogen through TLR/NFκB signaling cascade and Lysozyme. Three OpHeps would be potential targets for prevention of bacterial infections in aquaculture industry of spotted knifejaw, which provided a new idea for the molecular breeding of fish disease resistance.

Excessive Erythrophagocytosis Accounts for Systemic Inflammation in Chronic Kidney Disease.

Chronic kidney disease (CKD) is associated with persistent systemic inflammation. Reduced red blood cell (RBC) survival in patients with CKD has been identified for several decades. The purpose of this study is to explore whether excessive erythrophagocytosis exists and contributes to systemic inflammation in CKD. A CKD rat model was induced by 5/6 nephrectomy. Erythrocyte osmotic fragility was determined with hypotonic NaCl solutions. Erythrocyte deformability was evaluated by filterability. RBC cell death was quantified using fluorescence-activated cell sorting analyses of fluorescent annexin V-bound surface phosphatidylserine (PS). Erythrophagocytosis was evaluated in vivo and in vitro. RT-qPCR and immunohistochemistry were used to determine the inflammatory effects after erythrophagocytosis. Erythrocyte osmotic fragility and deformability progressively declined, and the percentage of PS-exposing RBCs progressively increased in CKD rats. Levels of erythrophagocytosis in vivo were evaluated by autologous injection of CFSE-labeled erythrocytes. In comparison with the control group, higher fluorescence intensity of CFSE was detected in the spleen homogenates of rats with CKD. In vitro, more of erythrocytes from 5/6Nx rats were phagocytosed by peritoneal macrophages in comparison to those from control rats. Compared with macrophages phagocytosed control erythrocytes, macrophages phagocytosed CKD erythrocytes exhibited higher mRNA levels of IL-6, CXCL-10, CXCL-11, iNOS, IL-1β, ICAM-1 and MCP-1. Compared with the control group, the red pulp of rats with CKD exhibited higher levels of p-NFκB, IL-6, iNOS and CXCL-10. ELISA results showed significantly increased plasma levels of both IL-6 and CXCL-10 in patients with long-term hemodialysis compared with those in healthy controls (2.30 ± 1.38 pg/mL vs 1.33 ± 0.65 pg/mL, P=0.01; 78.11 ± 27.34 pg/mL vs 37.45 ± 7.08 pg/mL, P=0.001). Our results indicated that excessive erythrophagocytosis may contribute to systemic inflammation in CKD.

Pharmacological inhibition reveals participation of the endocytic compartment in positive feedback IL-6 secretion in human skeletal myotubes

IL-6 is an important cytokine involved in metabolic, immunological, and cell-fate responses. It is released upon stimulation by skeletal muscle cells through partially characterized mechanisms. In some cell types, IL-6 has been reported to activate a positive feedback loop involving endocytic vesicles, but evidence is mostly based on transcription and signal transduction mechanisms and is very scarce in muscle cells. Our aim was to directly demonstrate the presence of positive feedback in the ATP-induced release of IL-6 into the supernatant of human skeletal muscle cultures. The total release (production) of IL-6 was reduced for higher volumes of supernatant, when the secreted IL-6 molecules are more diluted, and enhanced when the supernatant volume was lower. In addition, secretion was impaired both by tocilizumab, a blocker of human IL-6 receptors, and by the soluble form of the receptor. The secretion in response to ATP was also inhibited by treatment with the endocytosis inhibitor dynasore, and by disruption of the acidic gradient of the endocytic compartment using different methods (chloroquine, NH4Cl or monensin). IL-6 secretion was also impaired by NED-19, a specific inhibitor of the two pore channels receptor mediating Ca2+ release from the endolysosomal compartment. IL-6 and ATP increased IL-6 mRNA levels, an effect blocked by tocilizumab. Altogether, our results demonstrate that ATP-secreted IL-6 activates a positive loop based on IL-6 receptors, endocytosis, two pore channels and IL-6 transcription. Given the importance of muscle IL-6 as a systemic regulator and as an inflammatory mediator, our study can help to understand muscle pathophysiology.

Possible effects of ancestry-related oxysterol-binding protein-like 10 genetic polymorphisms on dengue virus replication and anti-dengue immune response

PurposeOxysterol-binding protein-like 10 (OSBPL10) gene has been associated with reduced susceptibility to severe dengue in individuals of African descent. The aim of this study was to determine the possible effect of OSBPL10 on dengue virus (DENV) replication as well as the impact of African and European haplotypes of six OSBPL10 small nuclear polymorphisms (SNPs) on dengue multiplication and innate immune response. MethodsWe conducted gene knockdown experiments targeting OSBPL10 in THP-1 and Huh-7D12 cell lines, followed by a DENV-2 replication assay. Extracellular viral load was determined using qRT-PCR. To investigate the impact of SNPs haplotypes on viral replication and gene expression we cultured peripheral blood mononuclear cells (PBMC) from individuals with homozygous African and European haplotypes of OSBPL10 with DENV-2. Individual genotyping was performed using High Resolution Melt (HRM) analysis. The level of viral replication was assessed through plaque assay, while RT-PCR was employed to determine the expression levels of RXR-α, IFN-γ, IL-10 and IL-8 genes. ResultsIn vitro OSBPL10 knockdown significantly reduced DENV-2 replication. Individuals carrying European haplotypes showed higher DENV titers along with elevated levels of RXR-α and IL-8 mRNA compared to those carrying African haplotypes, who exhibited lower viral loads alongside increased IFN-γ and IL-10 expression. ConclusionsOur findings further explore the role of OSBPL10 in DENV multiplication, immune response to infection. The European haplotypes of OSBPL10 appear to increase DENV replication and promote RXR-α and IL-8 mRNA expression which correlates with the suppressive effect of these mediators on type I IFN, promoting viral replication and a deficient antiviral response. In contrast, the African haplotype showed a reduction in DENV replication and enhanced IFN-γ and IL-10 mRNA expression, which could be related to the better management of dengue infection and the low frequency of severe disease in this ethnic groupe.

Novel mechanisms of intestinal flora regulation in high-altitude hypoxia

BackgroundThis study investigates the molecular mechanisms behind firmicutes-mediated macrophage (Mψ) polarization and glycolytic metabolic reprogramming through HIF-1α in response to intrinsic mucosal barrier injury induced by high-altitude hypoxia. MethodsEstablishing a hypoxia mouse model of high altitude, we utilized single-cell transcriptome sequencing to identify key cell types involved in regulating intestinal mucosal barrier damage caused by high-altitude hypoxia. Through proteomic analysis of colonic tissue Mψ and metabolomic analysis of Mψ metabolites, we determined crucial proteins and metabolic pathways influencing intestinal mucosal barrier damage induced by high-altitude hypoxia. Mechanistic validation was conducted using RAW264.7 Mψ in vitro by assessing cell viability with CCK-8 assay following treatment with different metabolites. The hypoxia mouse model was further validated in vivo by transplanting gut microbiota of Firmicutes. Histological examinations through H&E staining assessed colonic cell morphology and structure, while the FITC-dextran assay evaluated intestinal tissue permeability. Hypoxia probe signal intensity in mouse colonic tissue was assessed via metronidazole staining. Various experimental techniques, including flow cytometry, immunofluorescence, ELISA, Western blot, and RT-qPCR, were employed to study the impact of HIF-1α/glycolysis pathway and different gut microbiota metabolites on Mψ polarization. ResultsBioinformatics analysis revealed that single-cell transcriptomics identified Mψ as a key cell type, with their polarization pattern playing a crucial role in the intestinal mucosal barrier damage induced by high-altitude hypoxia. Proteomics combined with metabolomics analysis indicated that HIF-1α and the glycolytic pathway are pivotal proteins and signaling pathways in the intestinal mucosal barrier damage caused by high-altitude hypoxia. In vitro cell experiments demonstrated that activation of the glycolytic pathway by HIF-1α led to a significant upregulation of mRNA levels of IL-1β, IL-6, and TNFα while downregulating mRNA levels of IL-10 and TGFβ, thereby promoting M1 Mψ activation and inhibiting M2 Mψ polarization. Further mechanistic validation experiments revealed that the metabolite butyric acid from Firmicutes bacteria significantly downregulated the protein expression of HIF-1α, GCK, PFK, PKM, and LDH, thus inhibiting the HIF-1α/glycolytic pathway that suppresses M1 Mψ and activates M2 Mψ, consequently alleviating the hypoxic symptoms in RAW264.7 cells. Subsequent animal experiments confirmed that Firmicutes bacteria inhibited the HIF-1α/glycolytic pathway to modulate Mψ polarization, thereby mitigating intestinal mucosal barrier damage in high-altitude hypoxic mice. ConclusionThe study reveals that firmicutes, through the inhibition of the HIF-1α/glycolysis pathway, mitigate Mψ polarization, thereby alleviating intrinsic mucosal barrier injury in high-altitude hypoxia.

Endoplasmic Reticulum Stress Contributes to Intestinal Injury in Intrauterine Growth Restriction Newborn Piglets.

Intrauterine growth retardation (IUGR) in piglets is associated with a high rate of morbidity and mortality after birth due to gut dysfunction, and the underlying mechanisms remain poorly understood. This study selected six pairs of IUGR newborn male piglets and normal birth weight newborn piglets (Large White × Landrace) to investigate differences in intestinal structure and digestive functions, intestinal ERS and apoptosis, intestinal barrier function, and inflammatory response. The results showed that IUGR significantly reduced the jejunal villi height (p < 0.05) and the ratio of villus-height-to-crypt-depth (p = 0.05) in neonatal piglets. Additionally, the microvilli in the jejunum of IUGR neonatal piglets were shorter than those in normal-weight piglets, and swelling of the mitochondria and expansion of the endoplasmic reticulum were observed. IUGR also significantly reduced serum glucose and lactase levels (p < 0.05) while significantly increasing mRNA levels of jejunal IRE1α, EIF2α, CHOP, Bax, Caspase9, Mucin2, Claudin-1, Occludin, ZO-1, Bcl-2, IL-6, and IFN-γ (p < 0.05), as well as GRP78 protein levels in neonatal piglets (p < 0.05). These findings suggest that IUGR impairs intestinal structure and barrier function in newborn piglets by enhancing intestinal inflammatory responses, activating intestinal ERS and the signaling pathways related to the unfolded protein response, thereby inducing ERS-related apoptosis.

Improvement of androgenic alopecia by extracellular vesicles secreted from hyaluronic acid-stimulated induced mesenchymal stem cells

BackgroundAndrogenetic alopecia (AGA) is a common form of hair loss. Androgens, such as testosterone and dihydrotestosterone, are the main causes of AGA. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can reduce AGA. However, preparing therapeutic doses of MSCs for clinical use is challenging. Induced pluripotent stem cell-derived MSCs (iMSCs) are homogenous and easily expandable, enabling scalable production of EVs. Hyaluronic acid (HA) can exert various functions including free radical scavenging, immune regulation, and cell migration. Herein, we examined whether hyaluronic acid (HA) stimulation of iMSCs could produce EVs with enhanced therapeutic outcomes for AGA.MethodsEVs were collected from iMSCs primed with HA (HA–iMSC–EVs) or without HA (iMSC–EVs). The characteristics of EVs were examined using dynamic light scattering, cryo-transmission electron microscopy, immunoblotting, flow cytometry, and proteomic analysis. In vitro, we compared the potential of EVs in stimulating the survival of hair follicle dermal papilla cells undergoing testosterone-mediated AGA. Additionally, the expression of androgen receptor (AR) and relevant growth factors as well as key proteins of Wnt/β-catenin signaling pathway (β-catenin and phosphorylated GSK3β) was analyzed. Subsequently, AGA was induced in male C57/BL6 mice by testosterone administration, followed by repeated injections of iMSC–EVs, HA–iMSC–EVs, finasteride, or vehicle. Several parameters including hair growth, anagen phase ratio, reactivation of Wnt/β-catenin pathway, and AR expression was examined using qPCR, immunoblotting, and immunofluorescence analysis.ResultsBoth types of EVs showed typical characteristics for EVs, such as size distribution, markers, and surface protein expression. In hair follicle dermal papilla cells, the mRNA levels of AR, TGF-β, and IL-6 increased by testosterone was blocked by HA–iMSC–EVs, which also contributed to the augmented expression of trophic genes related to hair regrowth. However, no notable changes were observed in the iMSC–EVs. Re-activation of Wnt/β-catenin was observed in HA–iMSC–EVs but not in iMSC–EVs, as shown by β-catenin stabilization and an increase in phosphorylated GSK3β. Restoration of hair growth was more significant in HA–iMSC–EVs than in iMSC–EVs, and was comparable to that in mice treated with finasteride. Consistently, the decreased anagen ratio induced by testosterone was reversed by HA–iMSC–EVs, but not by iMSC–EVs. An increased expression of hair follicular β-catenin protein, as well as the reduction of AR was observed in the skin tissue of AGA mice receiving HA–iMSC–EVs, but not in those treated with iMSC–EVs.ConclusionsOur results suggest that HA–iMSC–EVs have potential to improve AGA by regulating growth factors/cytokines and stimulating AR-related Wnt/β-catenin signaling.

Deubiquitinase USP14 is upregulated in Crohn's disease and inhibits the NOD2 pathway mediated inflammatory response in vitro.

The nucleotide binding oligomerization domain containing 2 (NOD2) protein and its ligand N-acetyl muramyl dipeptide (MDP) are crucially involved in Crohn's disease (CD). However, the mechanism by which NOD2 signaling is regulated in CD patients remains unclear. Ubiquitin specific protease (USP14) is a deubiquitylase that plays an important role in immunity. This study aimed to investigate the mechanism by which UPS14 regulates NOD2 induced inflammatory response in CD and inflammatory bowel diseases (IBD). Our results showed that USP14 protein and mRNA levels in intestinal tissues of CD patients were significantly higher than those in healthy controls. In addition, USP14 was upregulated in IBD mouse model. While treatment with MDP, TNF-α or the Toll-like receptor 1/2 agonist Pam3CSK4 all led to significantly higher mRNA levels of TNF-α, IL-8 and IL-1β in THP-1 cells, pretreatment with USP14 inhibitor IU1 could stimulate further upregulation of TNF-α, IL-8 and IL-1β. In particular, MDP promoted the activation of JNK, ERK1/2 and p38 as well as NF-kB in THP-1 cells, and IU1 significantly enhanced the MDP-induced activation of these proteins without effects on USP14 protein level. Furthermore, the JNK inhibitor sp600125, ERK1/2 inhibitor U0126 or P38 MAPK inhibitor PD169316 significantly decreased the mRNA levels of TNF-α, IL-8 and IL-1β in THP-1 cells stimulated by both IU1 and MDP. In conclusion, our findings suggest that USP14 could inhibit MDP-induced activation of MAPK signaling and the inflammation response involved in IBD, and that USP14 is a potential therapeutic target for IBD.